With more than 800 members, G-protein-coupled receptors (GPCRs) represent the largest family of cell surface molecules involved in signal transmission, accounting for >2% of the total genes encoded by human genome. Members of the GPCR superfamily share a common membrane topology: an extracellular N-terminus, an intracellular C-terminus and seven transmembrane (TM) helices, which are connected by three intracellular loops and three extracellular loops. On the basis of their shared topological structure, GPCRs are also referred to as seven transmembrane (7TM) receptors. These receptors control key physiological functions, including neurotransmission, hormone and enzyme release from endocrine and exocrine glands, immune responses, cardiac- and smooth-muscle contraction and blood pressure regulation. Their dysfunction contributes to some of the most prevalent human diseases. Emerging experimental and clinical data indicate that GPCRs have a crucial role in cancer progression and metastasis. Hence, there is the possibility that some GPCRs may be suitable targets for anti-cancer drugs.
Chemokines play an important role inter alia in immune and inflammatory responses in various diseases and disorders, including cancer, viral infections, asthma and allergic diseases, as well as autoimmune pathologies such as rheumatoid arthritis and atherosclerosis. Depending on their structure, chemokines are classified as C-C chemokines (containing a cysteine-cysteine motif) or C-X-C chemokines (containing a cysteine-X-cysteine motif). Receptors that bind such chemokines are thus classified as members of the CCR or CXCR family, respectively.
CXCR4 (also called fusin, HM89, LESTR, HUMSTR) is an alpha-chemokine receptor specific for stromal-derived-factor-1 (SDF-1, also called CXCL12 and PBSF), a molecule endowed with potent chemotactic activity for lymphocytes. This receptor is one of several chemokine receptors that HIV isolates can use to infect CD4+ T cells. Several normal tissues express CXCR4. Notable examples of cells where expression has been demonstrated, both in terms of mRNA and the functional protein, include: hematopoietic cells/bone marrow progenitor cells; cells of the immune system, e.g. T cells, pre-B and plasma cells, dendritic cells, NK cells; other blood cells, e.g. monocytes, mast cells, platelets; cells of the nervous system, e.g. neurons, astrocytes, microglia; other cells, e.g. endothelial cells, vascular smooth muscle, gastrointestinal epithelium, and certain other epithelial cells.
CXCR4 is expressed in a variety of tumours and plays a decisive role in the pathophysiology of cancer, particularly in cancer metastasis (Dorsam and Gutkind, 2007, G-protein-coupled receptors and cancer. Nat Rev Cancer 7: 79-94). CXCR4 expression has been found in almost all tumours studied. It is also of interest that SDF-1 is expressed at particularly high levels in liver, lung, bone marrow, lymph nodes, and (at somewhat lower levels) in brain, i.e. sites to which cancers typically metastasize. Potential indications for an agent such as an antibody targeting CXCR4 include cancers (e.g. metastatic cancers), e.g. of breast, prostate, pancreas, esophagus, colorectal, liver and lung (both SCLC and NSCLC), as well as malignant or metastatic melanoma, brain tumours (glioma), head-and-neck cancers, certain leukemias, lymphomas such as non-Hodgkins lymphoma, childhood tumours (e.g. neuroblastoma), renal cancer, hemangioblastoma. Overexpressed CXCR4 has been found in several other cancers, including lung tumours, non-small cell lung cancer, ovarian cancer, cervical cancer, papillary thyroid carcinomas, osteosarcomas, and other malignancies. An anti-CXCR4 antibody can also be used for treatment of viral infections such as HIV or other retroviral infections, and in the treatment of immune diseases such as autoimmune diseases, inflammatory diseases and in the inhibition of angiogenesis and vascularization.
Due to their complex structures, GPCRs are considered as “difficult targets” for raising specific antibodies. They can neither be easily purified from the membrane fraction of lysed cells, nor be recombinantly produced in different expression systems as correctly folded soluble proteins.
The difficulties associated with generating antibodies against GPCRs are set out in Hoogenboom et al. Eur. J. Biochem 260, 774-784 (1999). Furthermore, Sui et al. Eur. J. Biochem 270, 4497-4506 (2003) explain the difficulties associated with trying to obtain human antibodies against the GPCR chemokine receptor CXCR4 and report that even using the pathfinder method combined with step-back selection no specific antibodies could be identified. Thus, in the field of GPCRs, the generation of specific antibodies remains a major challenge.
Northwest Biotherapeutics Inc. is developing monoclonal antibodies against CXCR4, some of which are in late stage preclinical development. Data suggesting the potential efficacy of CXCR4 antibodies were gathered in animal models by NWB and others. Subsequent development may include humanization of selected antibodies and toxicity studies in preparation for Phase I clinical trials.
MDX-1338 is an anti-human CXCR4-specific, fully human monoclonal antibody from Medarex. In vitro studies demonstrated that MDX-1338 binds to CXCR4-expressing cells with low nanomolar affinity. MDX-1338 blocks CXCL12 ligand binding to CXCR4 expressing cells and inhibits CXCL12 induced migration and calcium flux with low nanomolar EC50 values. MDX-1338 is an IgG4, and thus lacks ADCC and CDC activity. MDX-1338 induces apoptosis in a range of CXCR4 expressing cell lines and also has anti-tumor activity in multiple AML and lymphoma tumor xenograft models.
In addition, there are a number of small molecule compounds and peptides at different stages of development that target CXCR4/SDF-1.
The inventors have recognized that the identification of additional antibodies that recognize CXCR4 would be of benefit in expanding the number of therapeutic options. As discussed above however, the nature of GPCRs such as CXCR4 means that the development of such antibodies poses real challenges.
In particular, there is a need for human antibodies to CXCR4 which recognise CXCR4 in its native membrane bound form. Although human antibodies are generally recognized to display advantages, it is known that the development of human antibodies that have high enough affinities and appropriate functional properties to make them candidates for successful human therapy is by no means straightforward. This is even more so the case with GPCRs, due to their complex and transmembrane nature.